S-Adenosyl-L-methionine, known as SAMe, is the main biological donor of methyl groups and it has several important therapeutic applications. As a substance existing in the living body, SAMe has been found to possess various pharmacological actions such as improvement of energy state of ischemic brain, improvement of cerebral energy metabolism and acidosis of the model with recirculated blood flow following ischemia, etc. Variety of other functions such as inhibition of neuronal death following ischemia, improvement of cerebral glucose utility, inhibition of brain edema, improvement of EEG, improvement of evoked potential, amebiorative action on motor function, and therefore reported to be important as a cure for stroke. SAMe as an antioxidant, use for osteoarthritis, liver protection and to control aging in elderly people is also suggested SAMe is an important molecule in normal cell function and its survival. SAMe is utilized by three key metabolic pathways: trans-methylation, trans-sulfuration and polyamine synthesis. In transmethylation reactions, the methyl group of SAMe is donated to a large variety of acceptor substrates including DNA, phospholipids and proteins. In trans-sulfuration, the sulfuration of SAMe is converted via a series of enzymic steps to cysteine, a precursor of taurine and glutathione, a major cellular anti-oxidant. Given the importance of SAMe in tissue function, it is not surprising that this molecule is being investigated as a possible therapeutic agent for the treatment of various clinical disorders as mentioned in Int. J. Biochem. Cell Biol. (2000), 32(4), 391-395.
There are numerous methods known to prepare SAMe at various scales and all are enzymatic and fermentation based. JP 58036397, JP 60070097, JP 56099499 and JP 54154774 describe the preparation of S-adenosyl-L-methionine using yeast. In this process the yeast extract was adsorbed on the resin and SAMe was eluted using suitable acids. The dilute solution of the product is concentrated using reverse osmosis and the product was isolated by spray-drying. Alternatively in RO 63045, CA 1057681 and DE 2530898, use of picrolinic acid was suggested for the product isolation from the fermented mass.
Use of Saccharomyces cultured on methionine media, cells of Rhizopus pseudochinesis cultured in a medium containing methionine and the use of different cultures of various origin are reported in JP 48044491, JP 47037038, JP 53005399, and JP 50082288. Microbial production of S-adenosyl-L-methionine by reacting adenosine triphosphate (ATP) and methionine catalyzed by enzyme from yeast or other fungi and the Lactobacillus bulgaricus containing the yeast extract are described in JP 57099199, JP 57086297 and JP 57086298.
In all the above methods, enrichment of (S,S)-isomer of SAMe has been achieved; however, it is not exclusive. Normally, percentage observed for the (S,S)-isomer in the SAMe samples analyzed by HPLC method was ranging from 60% to 75%. The varying isomer ratios are attributed to the method of product isolation and the temperature at which the enzyme reaction is effected.
All the above methods have several limitations with respect to the productivity per day and require high investment. Some of the problems associated with these methods are as under:    1. Isolation of required enzyme from its natural sources is difficult and for few milligrams of enzyme a large quantity of cells is required.    2. Enzymatic synthesis of SAMe indicated the problem of product inhibition. The 5 and 10 mM reactions do not even form 1 mM of SAMe. The same is the case with immobilized enzymes. Thus, in enzymatic synthesis, non-competitive product inhibition of SAMe vs methionine leads to decrease in the rate of SAMe production at high concentration as reported in the Biotechnol. Appl. Biochem. (1987), 9(1), 39-52.    3. The product isolation is tedious and various techniques like ultra-filtration with molecular cut off, ion exchange resins columns and reverse osmosis need to be used. Thus, it requires high investment to adopt the above methods, apart from the limitations due to heavy reactor occupancy and very high dilution involved during downstream processing.
Thus the prior art teaches the production of SAMe by fermentation. While there are a few stray attempts to synthesize SAMe chemically, they have met with little success for manufacture of SAMe on commercial scale. The reason being that chemical method does not normally give the required minimum enrichment of (S,S)-isomer wherein (R,S)-isomer is 55-65% and the required (S,S)-isomer is 35-45%. The available methods produce a lot of side products owing to the presence of multiple centers in S-adenosyl-L-homocysteine susceptible to methylation.
A report by Jose R. Matos et al. published in the Biotechnol. App. Biochem. (1987), 9(1), 39-52. reveals the use of methyl iodide and trimethylsulfonium iodide (TMSI) for methylation of S-adenosyl-L-homocysteine and reports the formation of inactive isomer as a major product in a 60:40 [(R,S)-isomer:(S,S)-isomer] mole ratio. The reaction of methyl iodide was performed in 85% formic acid and was kept in dark for 3-5 days to complete. The product was isolated using Amberlite IRC-50 resin columns and lyophilized. The methylation reaction with TMSI has the disadvantage of demethylation as the concentration of dimethyl sulfide is increased in the reaction. At certain stage, reaction attains equilibrium and the formation of side product predominates. Both the methods are not useful for large scale manufacture due to its asymmetrically non-specific approach, longer reaction time, formation of side products and low yields of the required isomer. In addition, the quantum of the required isomer is much less than that obtained by the fermentation methods.
In our co-pending application we have reported the first-ever chemical process for the industrial manufacture of S-adenosyl-L-methionine with the enrichment of active (S,S)-isomer using trimethyloxonium tetrafluoroborate (TMOTFB) as a methylating agent, whose production involves the use of dimethyl ether gas, which is highly flammable gas and hence requires investment to handle in commercial scale production. Though this application teaches several methylating agent, this application is exemplified only with TMOTFB, and this application do not teach or suggest about the methylating agent of the present invention.
We have continued to our research to identify alternative methylating agent for methylation of S-adenosylhomocysteine which should be high yielding, reproducible on larger scale with the predominance of the active (S,S)-isomer and succeeded in identifying methylating agent which avoids hazardous gases like dimethyl ether.